Progenitor cells have multi-lineage potential when differentiated and therefore potentially lend themselves to a diverse range of therapeutic treatments. It is conceived that an enriched fraction of these cells delivered to a diseased or damaged tissue site may have maximum therapeutic benefit through accelerated tissue healing. Progenitor cells are typically a very rare population of cells (Caplan 2005) contained within a heterogeneous mixture of non-therapeutic cells in some mammalian tissues and fluids.
Various techniques and devices to capture cells have been reported in the literature including sedimentation (Takazawa & Tokashiki, 1989), centrifugation (Apelman et al, 1992; Jaeger, 1992) spin filters (Himmerlflab et al 1969) and cross-flow microfiltration (Maiorella et al 1991). However, these are associated with a number of disadvantages. Sedimentation processes suffer from low sedimentation velocity of cells and a slow separation process. Centrifugation devices rely on a radial force to drive cells outwards from the fluid and although high recovery is quickly achieved these devices typically have a high capital cost associated with them. In addition, high shear stresses could potentially damage cells or induce unwanted changes in e.g. progenitor cells. Spin filters and cross-flow filters use membranes to filter the cell suspension and these are susceptible to fouling with subsequent loss in performance. Other processes using such filters include controlled shear filtration (Vogel and Kroner, 1999), tangential flow filtration (Radlett, 1972) and dynamic filtration. The rate and efficiency of separation is dependent on the degree of fouling. Dynamic filtration relies on relative motion between the membrane and the housing to generate a shear flow independent of flow across the filter (Castilho & Medrohno, 2002). Vortex flow filters and rotating disc filters are the two common types of dynamic filtration devices (Stromberg et al, 1989). Karumanchi et. al. (2002) have provided a review of field-assisted separation techniques for cells and biomacromolecules which include electric, magnetic and acoustic phenomena to achieve the desired separation. Dielectrophoresis is the lateral motion of uncharged particles due to the polarization effect produced by non-uniform electric fields (Docoslis et al, 1997, 1999). This is applicable only to very low electrical conductivity media otherwise excessive heating of the fluid will occur. Magnetic separations fall into two types. In the first, the cells to be separated have an intrinsic magnetic property (eg. red blood cells, magnetotactic bacteria) and in the second type the non-magnetic component of the mixture that are required must be rendered magnetic by a magnetic-responsive entity. In both cases the fluid must be subjected to a magnetic field to achieve separation. Magnetic separation has been used extensively for numerous cell separations including bone marrow processing to enrich for progenitor cells (Roath et al, 1990). Acoustic separators have been widely used to separate micro-organisms and cells but almost universally rely on using ultrasonic waves. Ultrasonic cell retention is achieved due to the formation of standing waves when an ultrasonic wave is reflected in the direction opposite to the propagation of the wave. Cells become trapped in the pressure node planes of the standing wave. Kilburn et al, (1989) and WO95/001214, first reported the possibility of using these waves as a means to separate cells from suspension and many other reports using this technique have been reported since (Coakley et al, 1994; Dobelhoof-Dier, 1994; Gaida et al, 1996; Gorenflo et al, 2003, 2004). Ultrasonic separators require careful tuning and typically have a limited loading capacity whereby only a small volume of cells is recovered. In addition this technique is used to remove a homogeneous population of cells from the fluid phase and therefore cannot capture a specific population from a heterogeneous mixture.
The present invention is directed towards apparatus and methods for separating a solid fraction from a fluid sample. In particular the present invention is directed to the separation of a cell fraction from a heterogeneous mixture of cells within a fluid sample. The process is a single step process with the cell separation being based upon the mechanical filtration of a sample using only physical means.